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Clin Chem Lab Med 2005;43(7):735–740
2005 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2005.125

Determination of the antioxidant capacity in blood

Marc A. J. G. Fischer1,*, Theo J. M. Gransier2, of oxidative stress in various (patho)physiological

Lenie M. G. Beckers2, Otto Bekers2, Aalt Bast1 processes such as several pulmonary and cardiovas-
and Guido R. M. M. Haenen1 cular diseases and diabetes (2–4). This has boosted
1 research on these processes and on intervention in
Department of Pharmacology and Toxicology,
these processes with antioxidants. As a consequence,
Faculty of Medicine, Maastricht University,
there is an increased demand for adequate methods
Maastricht, The Netherlands
2
to monitor antioxidant protection.
Department of Clinical Chemistry, University
Antioxidants do not act in isolation; together with
Hospital Maastricht, Maastricht, The Netherlands other antioxidants, they form an intricate network. For
example, ascorbate can recycle oxidized vitamin E
Abstract and oxidized ascorbate can be recycled by gluta-
thione, as demonstrated by the pioneering work of
Background: A vast amount of scientific research is Hopkins and Evans (5) and of Slater and coworkers
directed towards the beneficial effects of antioxidants (6). To assess the total network of antioxidants, sev-
on health. For this reason, several assays have been eral strategies have been developed (7–10). In the
developed to determine the total antioxidant capacity
present study one of these strategies is evaluated, i.e,
of blood. Methods: In this study two procedures
the strategy based on the ability of an antioxidant to
based on the use of the green-blue 2,29-azino-bis(3-
scavenge 2,29-azinobis(3-ethylbenzthiazoline-6-sulfon-
ethylbenzthiazoline-6-sulfonic acid) radical (ABTS•q)
ic acid) (ABTS) radicals (ABTS•q). 6-Hydroxy-2,5,7,8-
were compared. In the first (commercially available)
tetramethylchroman 2-carbonic acid (Trolox)
procedure, ABTS•q was generated in the presence of
solutions are used to calibrate the antioxidant capac-
the blood sample. In the second procedure, referred
ity. Therefore, these assays are often referred to as
to as the decolorization assay, antioxidants react with
Trolox equivalent antioxidant capacity (TEAC) assays.
preformed ABTS•q. Results: It was found that the first
Scavenging of the non-physiological ABTS•q has
procedure leads to greater underestimation of the
been used as a tool to predict in vivo antioxidant
actual antioxidant capacity and is more prone to arti-
capacity.
facts than the second procedure. Therefore, only the
Initial TEAC assays involved the peroxidase activity
latter procedure was evaluated in detail and it
of heme-containing proteins to convert the colorless
appeared that (i) plasma is preferred over serum, (ii)
ABTS into the blue-green-colored ABTS•q (11). Origi-
the high background produced by albumin can be cir-
nally, this activity was used to determine the hemo-
cumvented by deproteination, (iii) samples can be
globin content of tissue samples by assaying
stored at y808C for 12 months, and (iv) the assay has
high precision. Due to poor linearity, the procedure formation of the green-colored radical (12). In this
has to be standardized to allow sample comparison. hemoglobin assay, reductive compounds such as
Conclusions: The decolorization assay is a reliable vitamin C interfere, since they react with the blue-
and robust assay that can be applied routinely to pre- green ABTS•q, giving a colorless non-radical product.
dict the antioxidant capacity of blood. This disadvantage was put to use in the TEAC assay.
The amount of ABTS•q that can be scavenged by a
Keywords: antioxidant; 2,29-azino-bis(3-ethylbenzthia- sample, frequently blood plasma, reflects the capacity
zoline-6-sulfonic acid) (ABTS); capacity. of that sample to neutralize free radicals (11). In this
way, the capacity of the total non-enzymatic antioxi-
dant network in the sample is determined by meas-
Introduction uring the reduction of the blue-green color. A
commercially available assay, the total antioxidant
Aerobic life is associated with the formation of reac- status (TAS) assay, is based on the reduction of the
tive oxygen species such as superoxide anion radicals accumulation of ABTS•q formed by the peroxidase
and hydroxyl radicals (1). Usually, the production of activity of metmyoglobin in the presence of anti-
reactive oxygen species is balanced by antioxidants. oxidants.
In certain situations, however, this balance is dis- In a more recent modification of the assay, pre-
turbed in favor of production of the reactive species, formed ABTS•q is used (13, 14). In this procedure, the
a condition denoted as oxidative stress. A large body decolorization of an ABTS•q solution by a sample –
of evidence has unequivocally proven the pivotal role which reflects the amount of ABTS•q that has been
scavenged by antioxidants in the sample – is deter-
*Corresponding author: Marc A.J.G. Fischer, Department of mined after a fixed period of time. The aim of the
Pharmacology and Toxicology, Faculty of Medicine, present study was to compare the commercially avail-
Maastricht University, 6200 MD Maastricht, The Netherlands
Phone: q31-43-3881417, Fax: q31-43-3884149, able TAS assay to a decolorization-type TEAC assay
E-mail: m.fischer@farmaco.unimaas.nl and to evaluate the applicability.

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736 Fischer et al.: Antioxidant capacity in blood

Materials and methods In the TAS assay, ABTS•q is generated by the peroxidase
activity of metmyoglobin in the presence of the sample. The
Chemicals assay is started by the addition of metmyoglobin and
ABTS•q begins to form. The ABTS•q formed reacts with anti-
ABTS and uric acid were obtained from Sigma Chemical Co. oxidants present in the sample until the antioxidants are
(St. Louis, USA), 2,29-azino-bis(2-amidinopropane) dihydro- consumed, whereafter the ABTS•q begins to accumulate.
chloride (ABAP) was obtained from Brunschwig Chemicals The concentration of ABTS•q that has accumulated within a
(Amsterdam, The Netherlands), Trolox was obtained from fixed time period is used to quantify the antioxidant capacity
Aldrich Chemical Co. (Milwaukee, USA), and the Randox of the sample.
TAS kit was obtained from Randox Laboratories Ltd. (Crum- In the TEAC decolorization assay, ABTS•q is generated
lin, UK). NaH2PO4ØH2O, Na2HPO4Ø2H2O, trichloroacetic acid chemically. Here, the assay is started by addition of the sam-
(TCA) and NaOH were of analytical grade purity and were ple to a solution containing pre-formed ABTS•q. The anti-
obtained from Merck Biochemica (Darmstadt, Germany). oxidants react with the ABTS•q, resulting in the formation of
colorless products. The decrease in ABTS•q concentration in
a fixed time period, due to the reaction of ABTS•q with the
Blood plasma and serum antioxidants present in the sample, is used to quantify the
antioxidant capacity of the sample.
The study was conducted according to the guidelines of the
most recent Declaration of Helsinki and the local medical
ethics committee. Human blood was collected in BD Vacu- TAS assay
tainer Systems (Becton Dickinson, Plymouth, UK) containing Determination of the antioxidant capacity of non-deprotei-
EDTA for plasma or SST II Gel and Clot Activator for serum. nated plasma or serum with the TAS assay was carried out
Plasma was obtained by centrifugation (5 min, 1000=g, 48C). according to the instructions of the manufacturer (Randox
For serum, blood was allowed to clot at room temperature Laboratories) on a Cobas Mira analyzer (Radiometer, Copen-
for 20 min. Serum was obtained by centrifugation (5 min, hagen, Denmark). In short, ABTS solution and the sample
1000=g, 48C). Plasma and serum deproteination was carried were transferred to a thermostated cuvet in cycle 1 (cycle
out by adding to plasma or serum an equal volume of a 10% time is 25 s). In cycle 3 the metmyoglobin solution was add-
(w/v) TCA solution. Plasma and serum samples were placed ed. The increase in absorption at 600 nm in the sample in
on ice for 5 min to complete deproteination, followed by cen- cycle 10 vs. cycle 2 was calculated. The resulting value (DA)
trifugation (5 min, 14,000=g, 48C). was compared with DA values for the Trolox calibrator (con-
centration 1.6 mM Trolox) to calculate the TEAC.
Determination of the antioxidant capacity
TEAC decolorization assay
Determination of the total antioxidant capacity of blood was
performed using the ability of antioxidants to scavenge the The TEAC decolorization assay was carried out as described
blue-green-colored ABTS•q (Figure 1). Two different types of by van den Berg et al. (13) with some modifications. Briefly,
assays were used, the commercially available TAS assay and ABTS•q was produced by incubating a solution of 0.23 mM
the decolorization TEAC assay (13). ABTS and 2.3 mM ABAP in 100 mM sodium phosphate buf-

Figure 1 The reaction of antioxidants with radical ABTS•q.

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Fischer et al.: Antioxidant capacity in blood 737

fer, pH 7.4 at 708C until the absorption of the solution from the same person revealed that the TEAC of
reached 0.70"0.02 at 734 nm. During the experiments, the serum was 89"5.7% (ns3, ps0.04) of that of plasma.
solution was stored at 48C. The lower TEAC of serum is probably caused by a
The determination was carried out using a Cobas Mira loss of antioxidants during serum preparation. There-
analyzer (Radiometer). In short, in cycle 1 the ABTS•q solu-
fore, plasma is preferred over serum.
tion was transferred to a thermostated cuvet and preheated
For determination of the total antioxidant capacity
for three cycles at 378C. In cycle 4, the sample was added.
The reduction in absorption in the sample at 752 nm in cycle of plasma, two different assay principles are applied:
16 vs. cycle 3 was calculated and corrected for reduction in the TAS assay and the TEAC decolorization assay. In
the blank. In the blank, an equal volume of buffer was added the TAS assay, ABTS•q is enzymatically generated by
instead of sample. The duration of one cycle was 25 s. The metmyoglobin. The assay is started by the addition of
resulting value (DA) was compared with DA values for the metmyoglobin. The ABTS•q that is formed reacts with
synthetic antioxidant Trolox. A calibration curve was con- antioxidants present in the sample. Once the antiox-
structed several times (Figure 2) using Trolox calibrators idants are consumed, there is a buildup of ABTS•q.
(concentration range 0–0.4 mM Trolox in the cuvet). The The concentration of ABTS•q that accumulates within
slopes of the calibration curves analyzed on 16 different days
6 min depends on the concentration and scavenging
were compared and a ‘‘response factor’’ was obtained that
capacity of antioxidants in the sample. In the TEAC
appeared to be constant. This response factor (0.710 mMy1)
was used to calculate the TEAC value of the samples by mul- decolorization assay, the sample is added to pre-
tiplying the DA value of the sample by this response factor.

Stability of the sample after blood collection

Plasma was divided into three portions. One portion was
immediately deproteinated using 10% TCA and kept on ice.
A second portion was directly stored on ice. A third portion
was stored at room temperature. At different time points, an
aliquot of the three samples was taken and the TEAC value
was assessed.

Determination of within- and between-batch

precision of the TEAC decolorization assay
Three different human plasma samples were analyzed 60
times on 2 different days and 10 times on 6 different days.
The within- and between-batch precision was calculated.

Figure 2 Calibration curve for the TEAC decolorization

Results and discussion assay, showing the mean calibration curve (black solid line
d) and "2 SD (dotted lines s) (ns16, R2s0.9995).
The antioxidant capacity of blood plasma is often
determined by quantifying the amount of ABTS•q that
can be scavenged by the antioxidants present in
blood plasma (11). When plasma is added to a solu-
tion of ABTS•q, there is fast reduction of the absor-
bance at 734 nm, due to the conversion of the
blue-green ABTS•q into colorless non-radical products
by antioxidants present in the plasma. This fast reduc-
tion is followed by a more gradual decrease in the
absorbance over time (Figure 3). This biphasic reac-
tion pattern is due to the presence of antioxidants in
the plasma that react either quickly or slowly with
ABTS•q (15, 16). Trolox, the compound used for cali-
bration, is an example of an antioxidant that reacts
quickly with ABTS•q (Figure 3).
The reduction in absorbance after a 6 min incuba-
tion period is mostly applied in assessing the anti-
oxidant capacity. It should be realized that within
6 min the scavenging of ABTS•q by antioxidants in the
plasma is not fully complete; the absorbance at
734 nm still declines after 6 min (Figure 3). This indi- Figure 3 Decrease in absorption (734 nm, measured on
spectrophotometer) over time of deproteinated plasma,
cates that the value obtained according to this pro-
10 mM Trolox and blank in the decolorization TEAC assay.
tocol is an underestimate of the actual antioxidant TEAC is determined after 6 min. As shown by the curve
capacity (15, 16). obtained with deproteinated plasma, reaction of the antiox-
Comparing the reduction in ABTS•q concentration idants in deproteinated plasma with ABTS•q is not complete
induced within 6 min for plasma and serum samples at 6 min.

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738 Fischer et al.: Antioxidant capacity in blood

formed ABTS•q. The reduction in ABTS•q concentra- potential error and the underestimation of the anti-
tion after 6 min reflects the antioxidant capacity of oxidant capacity of slow-reacting antioxidants in the
blood plasma, identical to the example given in Figure TAS assay imply that the decolorization assay is pre-
3. Both the TAS assay and the decolorization assay ferred over the TAS assay for determination of the
were used to determine the antioxidant capacity of a TEAC of blood plasma. Therefore, we focussed the
series of blood samples from healthy volunteers. subsequent evaluation on the decolorization assay.
Non-deproteinated plasma was used for this compar- Deproteination of blood plasma drastically reduced
ison because the TAS assay protocol is based on non- the TEAC of blood plasma. The average TEAC of
deproteinated samples. blood plasma determined with the decolorization
It was found that the TEAC range determined in 100 assay dropped from 7.38"0.37 to 0.61"0.07 mM
healthy volunteers varied from 1.05 to 2.50 mM using (ns100, p-0.001) after deproteination. It has been
the TAS assay, and from 5.51 to 7.08 mM with the reported that albumin is a major contributor to the
decolorization assay (Figure 4). The TAS values were TEAC of protein-containing plasma (11, 17). The non-
approximately one-sixth of the decolorization values. protein TEAC is due to low-molecular-weight endog-
As discussed above, the decolorization assay gives an enous and alimentary compounds, such as uric acid,
underestimation of the antioxidant capacity (Figure 3). ascorbate and flavonoids (10, 11). The TEAC is often
Apparently, with the TAS assay the deviation of the used to investigate the effect of dietary antioxidants,
measured capacity from the actual capacity is even indicating that deproteinated plasma is preferred.
greater. This originates from the relatively low con- Thus, the high background level due to proteins is cir-
centration of ABTS•q during the 6 min incubation in cumvented. Deproteination, however, might also
the TAS assay. At the beginning of the assay, no result in the loss of antioxidants bound to proteins
ABTS•q is present. ABTS•q has to be formed enzy- (18). This should be taken into consideration when
matically and the ABTS•q that is generated will first investigating the effects of dietary antioxidants.
react with fast-reacting antioxidants. The opportunity Applying different volumes of deproteinated blood
for slow-reacting antioxidants to react with ABTS•q in plasma in the decolorization assay revealed that the
the TAS assay is less compared with that in the decol- response in this assay is not linearly related to the
orization assay. In the decolorization assay, an excess volume of deproteinated plasma in the assay (Figure
of ABTS•q is present when the incubation starts. 5) (16). For more than 25 mL of plasma, the decrease
Another drawback of the TAS assay is that com- in absorbance of ABTS•q was relatively low. Under
pounds may reduce the formation of ABTS•q by inhib- these conditions, the greater part of the ABTS•q is
iting the peroxidase activity of the heme-containing consumed. A low concentration of ABTS•q will reduce
protein, metmyoglobin (16). This would result in a too the relative amount of antioxidants that will react with
high value of the TEAC. Interestingly, three plasma ABTS•q within the 6 min time period of the assay. This
samples had a relatively high TEAC in the TAS assay, indicates that in the procedure applied the sample
while the TEAC in the decolorization assay did not
deviate from the other samples (Figure 4). Possibly,
the relatively high TEAC we observed in the three
samples in the TAS assay (Figure 4) is due to the pres-
ence of peroxidase inhibitors in these samples. This

Figure 5 Relationship between the volume of plasma used

in the assay and decolorization of the ABTS•q solution.
Figure 4 Difference plot for 100 plasma samples in which Human plasma was used undiluted or diluted two–six-fold
the TEAC was measured with the TAS method and the decol- prior to deproteination. After deproteinating the plasma
orization assay. The mean difference (solid line), 95% confi- samples, DA at 752 nm was assessed as described in Mate-
dence interval for the mean difference (mean"2SEM; — —), rials and methods using the decolorization TEAC assay
and the limits of agreement (mean"2SD; -----) are shown. (mean"SD, ns5).

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Fischer et al.: Antioxidant capacity in blood 739

change in the concentration of a certain antioxidant

in plasma, which provides only a small contribution
to the TEAC, might be missed.
The decolorization TEAC assay was successfully
applied in several studies. For example, in smokers,
the higher the number of cigarettes consumed, the
higher was the amount of 4-aminobiphenylhaemo-
globin (4-ABP-Hb) adducts, probably due to the higher
dose of noxious compounds from cigarettes that are
responsible for the formation of 4-ABP-Hb adducts
(20). In the same population of smokers, the higher
the TEAC, the lower was the amount of 4-ABP-Hb
adducts (20). TEAC probably reflects protection of the
body against the formation of these 4-ABP-Hb
adducts.
Recently, van den Berg et al. (21) studied the poten-
tial benefits of a high fruit and vegetable intake on
antioxidant status (the decolorization TEAC assay), on
markers of oxidative damage to lipids, proteins and
DNA (including F2-isoprostane determination and the
Figure 6 Stability of the sample after blood collection. Plas- Comet assay), and on functional markers of oxidative
ma was immediately deproteinated with TCA (final concen- stress (including nuclear transcription factor-kB acti-
tration 5% w/v) and stored on ice until analysis (s), plasma vation). Based on the observed within-subject varia-
was stored on ice and deproteinated with TCA just before
bility during the control period, it was concluded that
analysis (h) or plasma was stored at room temperature and
deproteinated just before analysis (=). the sensitivity of TEAC was the highest of all the
markers studied. This indicates that, despite the pre-
viously mentioned disadvantages, the decolorization
volume should not exceed 25 mL, and that within a TEAC assay might be useful in studies on oxidative
study the sample volume for all samples should be stress.
identical (19).
The TEAC of blood plasma appeared to be fairly
stable. The TEAC was not affected by the procedure
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